Plant breeding corresponds to the domestication of plant species for the benefit of humans to obtain food, feed and fiber of sufficient quality and quantity. Plant breeding is a very old occupation of mankind and only in the course of the 20th century has the practical knowledge received a scientific foundation. Plant breeding was originally based on selecting and propagating those plants that were outperforming in local selection fields. With the rediscovery of the genetic laws and the development of statistical tools plant breeding became based on knowledge of genetics and was technologically supported by methods such as doubled haploids (DH)—see e.g. Haploids in Crop Improvement II eds; Palmer C, Keller W, and Kasha K (2005) in: Biotechnology in Agriculture and Forestry 56 Eds; Nagata T, Lörz H, and Widholm J. Springer-Verlag Berlin Heidelberg New York, ISBN 3-500-22224-3—and molecular markers—see e.g. De Vienne ed. (2003) Molecular Markers in Plant Genetics and Biotechnology. Science publishers Inc. Enfield, N.H. USA. ISBN 1-57808-239-0.
Plant breeding delivers genetic concepts tailored to a specific environment which allows its exploitation in an economic manner. This objective of plant breeding is achieved through the efficient utilisation of genetic variation which exists within the germplasm of plant species. Such genetic concepts comprise combinations of genes which lead to a desirable phenotype in a particular environment. This means that the plant parts which are harvested are maximised in yield and quality, at the lowest possible cost required to grow the plants and harvest the product. When plant breeding is applied at a commercial level, seed production is also an important issue. Seed production aims at the multiplication of plants by means of sexual reproduction, in which the genetic composition is preserved.
In addition, the commercial seeds need to be of sufficient quality to allow efficient germination. The preservation of genetic constitution through sexual reproduction is however a paradox, because sexual reproduction fundamentally exists to create offspring with new combinations of alleles. The genetic mechanisms that act during sexual reproduction have evolved to increase genetic variation, in order to enhance the chances of survival of a species in a changing environment. Meiotic recombination, independent chromosome assortment and the mating system are main contributing factors in this respect. Uniformity in offspring through sexual reproduction can therefore only be achieved when the parental plants are fully homozygous. Combining gametes of such plant will lead to the exact reproduction of the genetic composition of the parent in each subsequent generation.
In many crops, the commercial seeds result from a cross of two homozygous parental lines. This approach ensures that the F1 hybrid is heterozygous for several loci, which can result in hybrid vigour and uniformity. If a breeder wishes to improve an existing F1 hybrid variety or an inbred variety, he will traditionally need to make crosses and go through several rounds of empirical selection to achieve this objective.
As the knowledge of gene function in relation to plant growth and development is still limited, breeders still largely depend upon phenotypic selection. As during inbreeding many genes are in a heterozygous state, especially during early generations, the allelic variants of the genes responsible for the phenotypic value assigned to some of the individual plants can be lost easily. This is due to the fact that during sexual reproduction and inbreeding heterozygosity and specific gene interactions are lost. Therefore, in plant breeding these mechanisms may act counterproductively, especially in those cases where genetically heterozygous plants have been identified with high agronomic, horticultural or ornamental value. Sexual reproduction will result in the segregation of the desirable alleles.
Therefore there is a strong need for technology that efficiently allows the preservation of the genetic constitution during sexual reproduction of plants with a high agronomic, horticultural or ornamental value.
One possibility to perpetuate plants while preserving the genetic constitution is by vegetative propagation. This allows a complete preservation of the genetic composition, as multiplication occurs exclusively through mitosis. Plants have evolved natural mechanisms of vegetative propagation, which allow them to swiftly occupy habitats. For example, vegetative propagation can occur through the formation of tubers, bulbs or rhizomes. An alternative is to use in vitro or in vivo culture technology to produce cuttings. A commercial disadvantage of vegetative propagation technology, when compared to propagation through seeds, is the fact that it is labour-intensive and therefore costly. Furthermore, it is difficult to store plants for longer periods of time, which poses logistic problems, and the risks of infections of the plant material with pathogens like viruses is considerably larger as compared to a situation in which plant material is propagated through seeds.
Alternatively, vegetative propagation may be achieved through the formation of asexual seeds, which is generally referred to as apomixis. This phenomenon occurs naturally in a number of species, and it may be induced in sexually propagating plant species by genetic engineering. In theory, this can be achieved by making use of specific genes which naturally induce the three different steps of apomixis, i.e. apomeiosis, parthenogenesis and autonomous endosperm development. In practice, however, the genes responsible for the different steps have not yet been identified, and their interaction may be quite complicated.
On the other hand, artificial engineering of the apomixis components may be quite feasible. For example, by modifying different steps during meiosis it has been shown that meiosis can be essentially converted into mitosis. This so-called “MiMe approach” makes use of a combination of mutations which suppress double strand break formation (spo11-1), induce sister chromatid segregation during meiosis I (rec8) and skip the second meiotic cell division (osd1). Combining this approach with parthenogenesis and autonomous endosperm formation may ultimately result in engineered apomixis (d'Erfurth et al: Turning meiosis into mitosis; PLoS Biology 2009; WO/2010/079432). Although since long the potential of apomixis technology for plant breeding has been widely recognised, proof of concept is still not available.
As yet another alternative, use can be made of reverse breeding technology (WO03/017753). Reverse breeding is based on the suppression of meiotic recombination through genetic engineering or chemical interference, and the subsequent production of doubled haploid plants (DHs) derived from spores containing unrecombined parental chromosomes. These DHs differ with respect to their genetic composition solely as a consequence of the independent parental chromosome assortment which occurs during meiosis. Therefore, it is sufficient to make use of one co-dominant, polymorphic marker per chromosome to determine which of the DHs or lines derived thereof should be combined through crossing to reconstruct the genetic composition of the original starting plant. As such, application of reverse breeding technology allows genetic preservation of any fertile selected plant through seeds, even if its genetic composition is unknown.
A disadvantage of this technology is the fact that complete suppression of meiotic recombination results in the absence of chiasmata. This may lead to inappropriate chromosome segregation during meiosis I, which can lead to aneuploidy of the gametes and thus to reduced gamete viability and performance. When no chiasmata are formed during meiosis I, every chromosome has an independent 50% chance to move to either one of the poles. This means that the theoretical chance to make a spore with a full chromosome complement is (½)n, wherein n represents the haploid chromosome number. The frequency of balanced gametes therefore decreases with increasing haploid chromosome number. Although many crop species have a relatively low chromosome number (e.g. cucumber has 7 chromosomes per haploid genome; spinach has only 6) there are also economically important species with relatively high chromosome numbers. A good example is tomato, economically one of the most important vegetable crops, which has 12 chromosomes per haploid genome. This technical constraint significantly reduces the efficiency of reverse breeding technology.
As another alternative approach use can be made of plants regenerated from unreduced spores. This technology has been termed Near Reverse Breeding (WO2006/094773). The unreduced spores are formed preferentially as a consequence of the omission of the second meiotic division. This naturally occurring phenomenon is known as Second Division Restitution (SDR), and it can occur in plants during sexual reproduction concomitantly with regular meiotic events.
Near Reverse Breeding technology exploits SDR events by regenerating plants from unreduced spores, produced through natural or engineered SDR. Genes have been discovered which—when mutated—give rise to SDR, such as OSD1 and TAM1. The resulting plants (termed SDR-0 plants) are largely homozygous, and they can be subsequently used to produce traditional DHs. Molecular markers which are polymorphic between the paternal and maternal genomes of the starting plant can be used to identify those SDR-0 plants (and DHs derived thereof) that are largely complementary with respect to their genetic composition.
Crossing of these plants will result in the near-complete reconstruction of the genetic make-up of the original starting plant. However, due to meiotic recombination during the formation of the SDR-0 events and during the formation of the DHs derived thereof, the complementarity will not be complete. The reconstructed hybrids will genetically differ to some extent, both from each other and from the original starting hybrid plant. However, this variation will be strongly reduced when compared to a situation in which the DHs are derived directly from a regular meiotic event. Moreover, these DHs are genetically fixed, which means there is no room for further selection.
The advantage of integrating an SDR event in this process is that the selection for genetic complementarity occurs in a two-step process. The first step is concentrated on the proximal regions of the chromosomes, i.e. including the centromeres. The second step is directed towards the distal ends of the chromosomes, i.e. those regions which were exchanged due to recombination. This delayed genetic fixation reduces the complexity and increases the chances of finding largely complementary genotypes, especially when molecular markers are available for selection.
A further advantage of this approach is the fact that SDR can occur naturally during sexual reproduction and that it can be exploited as such without further need to interfere with sexual reproduction processes. Methods to further increase the normal prevalence of SDR events are known in the art, for example through stress treatment with N2O (as has previously been described in lily: Barba-Gonzalez et al (2006), Euphytica 148: 303-309; and in tulip: Okazaki et al. (2005), Euphytica 143: 101-114).
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.